The basic tenet of this proposal is that vaccines represent the most cost-effective medical intervention to prevent morbidity and mortality from infectious diseases. Worldwide, tuberculosis is the largest cause of death from a single infectious disease and although the PIs have long championed BCG as a vaccine against tuberculosis, in this competitive renewal application, they propose to develop effective and safe live attenuated tuberculosis vaccines by generating defined mutations in the M. tuberculosis genome in addition to generating defined mutations in the BCG genome. The PIs have developed a novel approach for mutagenizing or deleting biosynthetic genes in M. tuberculosis or BCG and for replacing the wild type gene by allelic exchange. Although BCG is the most widely used vaccine in the world, the ability to generate defined mutations will now allow the PIs to develop auxotrophic vaccines that are unable to cause disease, even in immunodeficient hosts. Secondly, the PIs propose to gain understanding of fundamental immunological mechanisms of protection against M. tuberculosis infection and for inducing pathogenicity and tissue damage by using selected gene-disrupted strains of mice and cellular approaches. Thirdly, the PIs propose a novel approach to identify protective antigens and open reading frames of any pathogen, which will facilitate development of recombinant BCG or attenuated M. tuberculosis vaccines able to protect against a variety of viral, bacterial and parasitic infections.